Human cytomegalovirus (HCMV) is the major cause of virus-induced congenital birth defects. It is also a major cause of pneumonitis, which is frequently fatal in immunocompromised adults. To better understand and eventually control this pathogen, we propose to continue our investigation of the genes and gene products that regulate HCMV gene expression and the cis-acting elements that respond to viral or cellular factors. This proposal focuses on several different viral gene products that regulate expression from the major immediate early (IE) enchancer containing promoter/regulatory region in the large unique component of the viral genome. These viral gene products are encoded by the tegument pp65 and/or the tegument pp71, the IE1 and the IE2 genes. The tegument protein pp65 and the IE1 protein up-regulate expression. In contrast, one or more of the IE2 proteins down-regulate expression. The IE2 transcription unit codes for a family of viral proteins. The IE2 proteins and functional domains that down-regulate the major IE promoter/regulatory region will be investigated. The IE2 proteins also activate, in trans, inducible viral promoters. IE2 proteins and functional domains that activate heterologous or homologous early or late viral promoters will also be investigated. The interaction of these viral proteins with eukaryotic cell transcription complexes will be investigated. The cis-acting elements in the major IE promoter/regulatory region that respond to cellular and viral transcription factors will be characterized. The first approach is to clone the viral genes and determine the effect of wild type or mutant viral proteins on regulation of the major IE promoter/regulatory region. A second approach is to identify the cis-acting element(s) by deletion analysis. Since the repeat elements are interspersed in the promoter/regulatory region, we will also synthesize each element and analyze its effects independently or in combination with other elements. The functions of these elements will then be tested independently or in combination. These experiments should contribute new information on how the eukaryotic cell and virus-specified proteins regulate viral gene expression for productive, persistent, or latent infection.